Ribosomal RNA

Transcription, Processing and Modification

rRNA constitute 80% of the RNA in rapidly dividing cell!

A growing mammalian cell must synthesize approximately10 million copies of each type of ribosomal RNA in eachcell generation to construct its 10 million ribosomes.

Eukaryotic ribosomes have four distinct rRNAs:

– Three in large subunit– One in small subunitIn humans– Large subunit contains – 28S, 5.8S and 5S– Small subunit contains – 18S

28S, 18S and 5.8S derived from the pre-rRNA5S from separate RNA – transcribed by RNA pol III

RNA POLYMERASE I - TRANSKRIPTS

Lokalisation: Nucleus:Transkripte: rRNA‘s

(außer 5S RNA)ITS.... internal

transcribed

spacer; 5.8S RNA: Homolog zum 5‘

Endeder 23S RNA (E.coli)

Lafontaine, D.L.J. and Tollervey, D., Nature Mol.Cell

Biol. 2 (2001), 514

200 rRNA

gene copies per haploide

genome,spread out in small clusters on five chromosomes. Transcription and processing takes place in the nuclear structure callednucleolus.

transcribed spacer DNA

The Nucleolus: Ribosome and RNP Factory - I

The Nucleolus: Ribosome and RNP Factory - II

rRNAs – synthesizing the precursor

A dark granule at the base of each fibril is a molecule of RNApolymerase

I with the newly synthesized transcript (fine thread) attached to it. At the speed of about 20 nt/s, over a thousand transcripts can be synthesized in an hour from a single gene.

Processing of Eukaryotic rRNAs

5'ETS 18SITS1

5.8SITS2

28S 3'ETS 5'ETS 18SITS1

5.8SITS2

3'ETS25S

A0 A1 D1A2

B1 EC2

B2C10 1 2 3 4 5 6

pseudouridylation 2'-O-methylation pseudouridylation 2'-O-methylation

47S 35Scut at 0, 6 cut at A0

45S 33Scut at 1 cut at A1, A2

41S 20S

27SA3

cut at 2

processing B1, B2

36Scut at 3

27SBprocessing

C1,C232S

cut at 4, 57S

processingE

18S 5.8S 28S

18S 25S5.8S

VERTEBRATES YEAST

A3

A

B

C

D

27SA2cut at A3

cut at D1

proteins involvedbelong to the familyof endonucleases,exonucleases,helicases, snoRNPproteins, exportfactors…

Pulse-Chase assays showing rRNA processing in yeast

Processing of Noncoding RNAs in the Nucleus

snoRNPs – small nucleolar ribonucleoproteinparticles

1. Prozessierung von rRNA2. 2‘-O-Methylierung von rRNA3. Pseudouridinylierung

(Ψ) von rRNA

Human: ~ 150 snoRNAs

in nucleolibox C+D snoRNAsbox H+ACA snoRNAs

E.coli: 4 2‘-O-Methylierungen, 10 ΨHuman: 106 2‘-O-Methylierungen, 91 Ψ

Clusters modifizierter Nukleotide

in den aktiven Stellen, aber nicht konserviert.Prokaryonten: keine snoRNA‘s, enzymat. Methylierungen

(Basenmethylierungen

vielhäufiger als in Eukaryonten).

Struktur der snoRNA-pre-rRNA

interaktion

ist wichtig für die Funktion.

Evolution: snoRNAs

zuerst nur rRNA

prozessieren dann Funktion der Modifizierungen.

snoRNAs come in the cell in the form of snoRNPs

proteins carrying enzymatic activity

snoRNP:box H+ACA: NAP57/Cbf5, Nhp2p, Nop10p

wichtig für die Stabilität, Gar1pfür Funktion (Gly/Arg

rich

domain: protein

snoRNP

Interaktion)box C+D: Nop58p: Stabilität, Nop56p und Nop1 (Fibrillarin, GAR Domäne oder RGG box): Funktion.

p

p p

TMG

PRNA pol II

ppp

PRNA pol III

PRNA pol II

E2RNA pol II

E1P

p

E3

p

p

E2RNA pol II

E1P E3

p p

En En+1

repeat 1 repeat 2 repeat n

A

B

C

D

Different strategies ofsnoRNA expression

their host genes belong to the 5’TOP family of vertebrate genes

Transcription – rRNA Processing

Role of small nucleolar RNAs (snoRNAs)

• snoRNPs associate with the rRNA before it is fully transcribed

• guide snoRNAs participate in nucleotide modifications (vast majority, also of other molecules than rRNAs) and pre-rRNA cleavage reactions (U3, U8, U13, U14, U22, U17, E2, E3, probably not directly involved in the catalysis)

…back to rRNA maturation…

5’ ETS

Hughes, J. M. X. J. Mol. Biol. 259, 645-654 (1996)

Proposed interaction of U3 snoRNA with the pre-rRNA

Smith, C. M. and Steitz, J. A. Cell 89, 669-672 (1997)

There are numerous snoRNAs involved in rRNA processing andmodification

Two peculiarities of pre-rRNA sequence:

Large numbers of methylated nucleotidesPsuedouridine residues

All modifications occur posttranscriptionallyAltered nucleotides at specific positionsClusteredAll altered nucleotides remain in final productSome unaltered nucleotide sections are discardedFunction of altered domains unclear

Examples of modified bases found in RNA

Dihydrouridine Pseudouridine 1-methylguanosine 7-methylguanosin

1-methyladenosine 2-thiocytidine 5-methylcytidine Ribothymine

pseudouridylationsugar methylation

Guide RNA-Mediated Modificationsof Precursor rRNAs

Kiss, T. Cell 109, 145-148 (2002).

snoRNAs act as guides for 2’-O-methylation and pseudouridylation

•The sites of modification depend on complementarity between the snoRNA andrRNA sequences

•snoRNA-directed modification can take place co-transcriptionally, thusfacilitating folding of the rRNA precursor

•Fibrillarin, a box C/D-associated protein, is most likely a 2’-O-methyl transferase

•Dyskerin/Cbf, a box H/ACA-associated protein, is a pseudouridine synthase

( )in yeast

( )in yeast

How do the guide-snoRNAs

guide?

Schematic structure of the guide snoRNAs

~100 of each class

Some unusual guides

•No single modification appears to be important, but globally they are believedto play a general role in RNA conformation and stabilization•Modifications tend to be concentrated in functional rRNA regions and to fine-tune ribosome activity and translation•Modifications are absent from regions where ribosomal proteins bind

The Nucleolus: Ribosomeand RNP Factory - III

Pseudoknot domain

CR4-CR5domain

CR7domain

H/ACA domain

H ACA

Template

IH1

Proposed secondary structure of vertebrate telomerase RNP

Telomerase is a specialized reverse transcriptase which provides the active site for RNA dependent DNA synthesis.

Germ cells contain telomerase.Somatic cells do not and the telomeres shorten with age.

Your life span may be determinedby the length of the telomeres atthe time of your birth.

Telomerase function

What is rRNA needed for?

To build the ribosomes (structural role)To help them function (catalytic role)

Ribosomes

Ribosomes - sites of protein synthesis

assembled in the nucleolus

exported into the cytoplasm

a. Free – unbound in the fluid cytoplasm, produce proteins for use in the cell

b. Bound – attached to the endoplasmic reticulum, produce proteins for export or for the plasma membrane

1. E. coli 70S model:

50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins

30S subunit = 16S (1,542 nt) + 20 proteins

2. Mammalian 80S model:

60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins

40S subunit = 18S (1,900 nt) + 35 proteins

Mammalian ribosome

Synthesis and processing of 5S rRNA

• Coded by a large number of genes outside of the nucleolus

• Transcribed by RNA polymerase III• 5’ end remains unchanged• 3’ end is truncated• Following synthesis 5S rRNA is transported to

nucleolus– Incorporated into assembly of ribosomes

Model of the 90S pre-ribosome

30S 50S

The central region of the interface side of the large subunit is

largely devoid of protein, and the nearest section of protein was found to be 18 Å

away from the peptide analogue bound at the peptidyl

transferase

centre. The region is entirely composed of tightly packed RNA from domain V of the 23

S rRNA. Since there is no way in which any protein could come close to

this site, the peptidyl

transfer must be an RNA-catalysed

reaction.

Ribosome is a ribozyme!